First described in snail neurons in 1982, voltage-activated proton-selective currents have been found in patch-clamp studies of several mammalian cells, including rat alveolar epithelium (DeCoursey, 1991) and human neutrophils (DeCoursey and Cherny, 1993). These H+ channels are opened by a combination of membrane depolarization, cytoplasmic acidification, and extracellular alkalinization. They appear to have been designed to extrude acid from cells. In phagocytes they are activated during the respiratory burst, the process of killing bacteria, thus they facilitate the inflammatory response. In general, proton channels serve to maintain homeostasis in cells during periods of high metabolic activity. Less is known about these channels than about most ion channels. The H+ channel molecule has not been positively identified, partly because there are no potent and specific inhibitors. One aim of this project is to characterize the inhibition of H+ channels by organic drugs, such as local anesthetics. The mechanism of interaction between organic compounds (e.g., local anesthetics or amantidine) and the channel molecule will reveal clues to the nature of the conduction pathway by which protons cross the membrane. One of the most potent and best-known inhibitors of H+ channels is Zn2+. We will explore the mechanism of the strong pH dependence of Zn2+ effects, and test the hypothesis that Zn2+ binds to distinct sites at the external and internal side of the channel, perhaps the same regulatory sites that control the exquisite pH sensitivity of the voltage dependence of gating (Cherny et al, 1995). We will use reagents that modify specific amino acids (histidine and cysteine) to determine whether these amino acids are exposed to the external or internal solution, and what roles they play in gating or permeation. The behavior of single H+ channels (which have not been detected previously) will be explored using several types of noise analysis as well as by direct measurement. Demonstration that H+ current fluctuations result from stochastic gating of H+ channels will provide strong evidence that the molecule is an ion channel rather than a carrier.